Malaria is a major threat to world health. Each year, there are 350-500 million clinical cases and greater than one million deaths due to malaria, primarily of children in sub- Saharan Africa. There is an urgent need for a safe and effective malaria vaccine and the development of such a vaccine is a global health priority. An abundance of data in animal models and in humans has indicated the existence of protective antigens and demonstrated that an effective vaccine against malaria is feasible. The recent sequencing and analysis Plasmodium genomes offer new tools to increase our understanding of parasite biology and lead to the development of new vaccines and drugs. However, novel high throughput approaches are needed to identify new targets for vaccine development. The goal of our proposed research is to identify new, immunogenic, pre-erythrocytic stage antigens for malaria vaccine development. The proposed research builds on the finding that immunization with radiation-attenuated sporozoites (RAS) provides high level (>90%) protection against challenge with infectious sporozoites of both the murine malaria parasite Plasmodium yoelii (Py) and the human malaria parasite Pf. The protection induced by RAS is due to the induction of CD8+ T cells targeting malaria antigens expressed by the pre-erythrocytic (sporozoite/liver) stages of the parasite life cycle. However, the majority of the antigen targets of these protective T cell responses induced by RAS are not known. Our hypothesis is that the P. yoelii antigens that are the targets of protective T cell responses induced by immunization with RAS can be identified using an in vitro system and such antigens will elicit strong protective immune responses when delivered as a vaccine. Furthermore, the corresponding P. falciparum orthologues will be excellent vaccine candidates. In this application, we propose to generate a defined array of adenovectors that express 300 highly expressed pre-erythrocytic stage genes (Specific Aim 1) and then screen the array to identify antigens that recall robust T cell responses from mice immunized with RAS (Specific Aim 2). Adenovectors from the array that recall the strongest T cell responses will be purified and evaluated for their ability to induce protective immune responses in mice (Specific Aim 3). P. falciparum orthologues of the protective antigens will represent high priority antigens for vaccine development. PUBLIC HEALTH RELEVANCE: There is an urgent need for a safe and effective malaria vaccine and the development of such a vaccine is a global health priority. Vaccines in clinical development, based on antigens that are currently known, fall short of the highly protective vaccine that is needed. The goal of this project is to discover new pre-erythrocytic malaria antigens, with high protective potential, which would be excellent candidates for inclusion in a malaria vaccine.